In order to maintain their shape and integrity, it is critical that all types of cells contain a structural scaffold. This structure is known as the cytoskeleton and is composed of a framework of interlocking proteins. The major protein component of the cytoskeleton is actin, and the assembly of actin monomers into the cytoskeleton is highly regulated.
Many cellular processes are mediated by the cytoskeleton. For example, changes occurring during cell cycle progression such as those associated with surface adhesion signals and the division of the cell into two daughter cells (mitosis) are dependent on the appropriate assembly and disassembly of the cytoskeleton. Therefore, it is currently believed that the survival of the cell depends on the controlled regulation of the cytoskeleton.
RhoB, a member of the Rho subfamily of small GTPases, is a protein that has been shown to be involved in a diverse set of signaling pathways including the regulation of the dynamic organization of the cytoskeleton during the cell cycle.
Within the cell cycle, RhoB is first detected at the G.sub.1 /S phase transition, reaching a maximal level during the S phase. In addition, RhoB has been localized to early endosomes and pre-lysosomal compartments within the cell. Since cytoskeletal modeling occurs during the S phase of the cell cycle and since RhoB is localized to membranous fractions, it has been suggested that RhoB plays a role in vesicular traffic or the translocation of factors necessary during the cell cycle (Zalcman et al., Oncogene, 1995, 10, 1935-1945). In support of this hypothesis are recent studies showing that, in Swiss 3T3 cells, RhoB recruits a protein kinase (PRK1) to endosomes (Mellor et al., J. Biol. Chem., 1998, 273, 4811-4814).
The RhoB gene has been classified as an immediate-early gene, which means that its transcription is rapidly activated upon exposure to certain growth factors or mitogens. The factors shown to activate RhoB transcription include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), genotoxic stress from UV light, alkylating xenobiotics and the retroviral oncogene v-fps. Each of these stimuli triggers DNA synthesis in cultures of high cell density (Engel et al., J. Biol. Chem., 1998, 273, 9921-9926). The response of RhoB to these factors implies a role for RhoB in wound repair and tissue regeneration upon growth factor stimulation and tumorigenesis upon mitogen stimulation.
Finally, manifestations of altered RhoB regulation also appear in disease states, including the development of cancer. Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer. Expression of constitutively activated forms of RhoB have been shown to cause tumorigenic transformation of NIH 3T3 and Ratl rodent fibroblasts (Khosravi-Far et al., Adv. Cancer Res., 1998, 72, 57-107). RhoB has also been shown to be overexpressed in human breast cancer tissues (Zalcman et al., Oncogene, 1995, 10, 1935-1945).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of RhoB. To date, strategies aimed at inhibiting RhoB function have involved the use of bacterial enzymes such as the Clostridium botulinum C3 exoenzyme which ADP ribosylates the protein rendering it inactive or agents (natural enzyme inhibitors) to inhibit the posttranslational modification (isoprenylation) of RhoB (Narumiya and Morii, Cell Signal, 1993, 5, 9-19). However, these targeting strategies are not specific to RhoB, as many proteins undergo similar posttranslational modifications. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting RhoB function.